5氮杂脱氧胞苷对鼻咽癌细胞RASSF1A基因甲基化和mRNA表达的影响

Effect of 5-Aza-CdR on methylation and mRNA expression of RASSF1A gene in nasopharyngeal carcinoma cells

目的:研究5氮杂脱氧胞苷对鼻咽癌CNE2细胞中RASSF1A基因甲基化和mRNA表达的影响。方法:用不同浓度(0、5、10、20μmol/L)5氮杂脱氧胞苷处理CNE2细胞,采用甲基化特异性PCR(MSP)法对处理后细胞中RASSF1A基因甲基化状态进行检测;并用SYBR Green qReal Time-PCR法检测RASSF1A mRNA的表达。结果:阴性对照组CNE2细胞中,RASSF1A基因呈完全甲基化状态,当用5μmol/L 5氮杂脱氧胞苷处理后,CNE2细胞出现非甲基化产物,20μmol/L 5氮杂脱氧胞苷处理后,CNE2细胞甲基化状态完全被逆转,均为非甲基化产物。阴性对照组CNE2细胞RASSF1A基因呈低表达,经5～20μmol/L 5氮杂脱氧胞苷处理后,RASSF1A mRNA的相对表达量逐渐增加,10和20μmol/L 5氮杂脱氧胞苷处理组,RASSF1A mRNA表达水平均明显高于阴性对照组(P<0.01);且20μmol/L处理组明显高于5μmol/L处理组(P<0.01)。结论:CNE2细胞RASSF1A基因甲基化可以被5氮杂脱氧胞苷逆转,且5氮杂脱氧胞苷可促进RASSF1A mRNA的表达。

OBJECTIVE：The relationship between methylation of RASSF1A gene and mRNA expression in nasopharyngeal carcinoma cell line CNE2 was investigated.METHODS：CNE2 cells were treated with various concentrations of 5-Aza-CdR.Methylation of RASSF1A gene was evaluated by methylation—specific polymerase chain reaction（MSP）.Expression of RASSF1A mRNA was detected by SYBR Green qRealTime-PCR.RESULTS：RASSF1A gene was completely methylated in the negative control group cell.De-methylation occurred in the 5μmol/L of 5-Aza-CdR. Methylation was completely reversed by 20μmol/L of 5-Aza-CdR.The expression of RASSF1A mRNA in the negative control group was low.The expression level increased gradually by 5,10 and 20μmol/L 5-Aza-CdR treatment. The levels in 10 and 20μmol/L treated group were significantly improved in comparison with the negative control group （P〈0.01）.RASSF1A mRNA in 20μmol/L treated group was obviously higher than that in 5μmol/L treated group （P〈0.01）.CONCLUSION：Methylation of RASSF1A gene in the nasopharyngeal carcinoma cell line CNE2 could be reversed by 5-Aza-CdR,promoting the expression of RASSF1A gene mRNA.